Fungicide consumption exacerbates the negative effects of a common gut parasite in bumble bee

Bumble bees face numerous environmental stressors, including gut-parasite infection and exposure to agricultural fungicides, which can negatively impact colony health. This study evaluates the interactive effects of these stressors on bumble bee (Bombus impatiens) microcolonies, focusing on colony development, worker survival, and parasite infection dynamics. Our aim in evaluating these interactions was to determine if bees would experience synergistic negative health outcomes compared to single stressor exposures. We reared forty queenless bumble bee microcolonies, and treated them with either fungicide-contaminated pollen, inoculation with a gut parasite, both, or neither. Contrary to original expectations, we did not observe significant synergistic interactions between the two stressors, however we found that consumption of fungicide was associated with higher likelihood of gut-parasite infection, and delayed recovery from infection. Fungicide consumption was also connected to smaller workers, and smaller male offspring. We also found that gut-parasite infection was correlated with decreased pollen consumption overall, decreased worker survival, and fewer developed pupae. This study provides insights into the impacts of co-occuring stressors affecting bumble bees and emphasizes the importance of sublethal effects on pollinator health.  

Methods
We established 40 queenless microcolonies of B. impatiens workers between June 9th and July 17th of 2022, and treated them with either fungicide-contaminated pollen, gut parasite inefction, both, or neither. Each microcolony consisted of 5 callow female workers.

To evaluate the effects of fungicide consumption and parasite infection, alone and in combination, on bumble bee microcolony growth and individual health, we established the following treatments. Starting 48 h after establishment, we fed half of our microcolonies (N = 20) 1 g pollen patties containing 15,000 ppb of the commercial fungicide Pristine® while the remaining microcolonies (N = 20) continued to receive untreated pollen. These pollen patties were replaced every 48 h, and we calculated pollen consumption following Runnion et al. (2024). Fourteen days after the establishment of each microcolony, we removed each worker from their microcolony and starved them for 1 h. We then randomly selected one worker from half of the fungicide-fed microcolonies (N = 10) and from half of the fungicide-free microcolonies (N = 10) and fed 1µl of 50/50 sucrose solution-Crithidia inoculum at a concentration of 600 Crithidia cells/µl (see electronic supplementary methods for inoculation details). Individuals from microcolonies not fed inoculum were instead fed 1µl of 50/50 sucrose solution. We recorded the identity of the inoculated bee and returned all workers to their microcolonies within 2 h of removal. This resulted in four stress treatments: control (-Fungicide, -Crithidia), fungicide only (+Fungicide, - Crithidia), parasite only (-Fungicide, + Crithidia), and combined (+Fungicide, + Crithidia). There were ten replicates of each treatment, blocked by spatial location in the rearing room. All workers within a block were sisters, sourced from the same commercial queenright colony.

Following treatment establishment, microcolonies were monitored daily for worker survival, time until first oviposition, and time until first offspring emergence.

Beginning 24 h after inoculation, we collected frass from all workers in the experiment for 14 days, in 24 h intervals. We extracted DNA from all frass samples. We conducted quantitative PCR using a Mastercycler® RealPlex2 (Eppendorf, Hamburg, Germany) for DNA extracted from frass samples.

Upon microcolony experiment completion, we placed each microcolony in a freezer until we later dissected each microcolony and recorded counts of total brood cells, honey pots, eggs, and dead and live larvae and/or pupae.

All analyses were completed in R.